Transcriptional downregulation of rice rpL32 gene under abiotic stress is associated with removal of transcription factors within the promoter region

Abstract

Background: The regulation of ribosomal proteins in plants under stress conditions has not been well studied. Although a few reports have shown stress-specific post-transcriptional and translational mechanisms involved in downregulation of ribosomal proteins yet stress-responsive transcriptional regulation of ribosomal proteins is largely unknown in plants.

Methodology/principal findings: In the present work, transcriptional regulation of genes encoding rice 60S ribosomal protein L32 (rpL32) in response to salt stress has been studied. Northern and RT-PCR analyses showed a significant downregulation of rpL32 transcripts under abiotic stress conditions in rice. Of the four rpL32 genes in rice genome, the gene on chromosome 8 (rpL32_8.1) showed a higher degree of stress-responsive downregulation in salt sensitive rice variety than in tolerant one and its expression reverted to its original level upon withdrawal of stress. The nuclear run-on and promoter:reporter assays revealed that the downregulation of this gene is transcriptional and originates within the promoter region. Using in vivo footprinting and electrophoretic mobility shift assay (EMSA), cis-elements in the promoter of rpL32_8.1 showing reduced binding to proteins in shoots of salt stressed rice seedlings were identified.

Conclusions: The present work is one of the few reports on study of stress downregulated genes. The data revealed that rpL32 gene is transcriptionally downregulated under abiotic stress in rice and that this transcriptional downregulation is associated with the removal of transcription factors from specific promoter elements.

Figure 1. Northern blot analysis of rpL32_8.1 under salt stress.

(A) Pokkali shoot (B) PB1 shoot (C) Pokkali root (D) PB1 root. t0.0 to t24.0 indicate the time of treatment (in h) with 200 mM NaCl ranging from 0 h to 24 h. The graphical representation of the data was deduced by normalizing the densitometric intensity of rpL32 genes on the blot with that of 28S rRNA in the corresponding lane of the EtBr gel.

Figure 2. Northern blot analysis of rpL32 genes located on chromosome 9 under salt stress.

(A) rpL32_9.3, (B) rpL32_9.2, (C) rpL32_9.1. t0.0 to t24.0 indicate the time after treatment of the Pokkali rice seedlings with 200 mM NaCl ranging from 0 to 24 h. All analyses were done as mentioned for Figure 1.

Figure 3. Nuclear Run-on analysis depicting the transcriptional activity of the rpL32 genes in shoots.

(A) Pattern of loading of different genes in the dot blots. The blots were hybridized with 3.45×10⁶ cpm/ml probe count. (B) Blot for control (no stress) condition. (C) Blot for salt stress (200 mM NaCl, 24 h) condition. (D) Graphical representation of the results of nuclear run-on assay. Densitometric quantification values for rpL32 genes were normalized with the densitometric readings obtained for 18S rRNA.

Figure 4. Northern blot analysis of rpL32_8.1 under various treatments and during stress recovery.

(A) Expression analysis under various treatments (sucrose, cold, drought and ABA) as mentioned below each lane. t0.0 to t24.0 indicate the time of treatment (in h). (B) Transcript abundance during stress recovery. t0 (lane 1) and t24 (lane 2) indicate the control (unstressed) and 24 h stressed (200 mM NaCl) tissues. t24-1 (lane 3), t24-15 (lane 4) and t24-24 (lane 5) indicate 1, 15 and 24 h of incubation during recovery from salt stress. All analyses were done as mentioned for Figure 1.

Figure 5. Promoter sequence analysis and transcription start site (TSS) mapping of rpL32_8.1.

(A) A ClustalW alignment of the japonica rice and indica rice Pokkali sequences corresponding to isolated genomic DNA portion. (B) Autoradiogram of denaturing urea polyacrylamide gel showing the three TSSs (TSS1 TSS2 and TSS3). Lanes T, A, C and G show the sequencing ladder of respective bases and the lane PE shows primer extension products. The TATA-box is marked with a shaded elliptical shape and the bands corresponding to the TSS are represented by elliptical borders. A portion of the DNA sequence downstream of TATA-Box is written besides the autoradiogram (as derived from the sequencing ladder) and the TSS bases are represented with bend arrows (showing the direction of the transcription). The distance of TSS1 (taken as +1) to the TATA-Box is also shown. (C) Schematic representation of the TATA-box and the observed TSS in the genomic DNA upstream of ATG codon of rpL32_8.1 gene. The underlined sequence represents the intron in the 5′UTR portion of the gene. The shaded arrow over the sequence represents the primer used in the primer extension. ATG start codon is boldly marked.

Figure 6. Analysis of rpL32_8.1 promoter for salt responsiveness.

(A) Schematic representation depicting the length of different promoter deletion fragments upstream of ATG start codon. (B) Agarose gel image showing PCR amplification of the different promoter fragments. (C) T-DNA portion of pCAMBIA1391z. BamHI and PstI sites were used for cloning of different promoter fragments. (D) – (I) Photographs of T1 generation transgenic seedlings showing GUS histochemical staining with X-Gluc. The different conditions and construct names are mentioned in each photograph. The wild type tobacco plant was used as negative control.

Figure 7. DMS-LMPCR in vivo footprinting of the promoter region of rpL32_8.1.

(A) In vivo footprinting of the top strand. Lane 1 shows the LMPCR products of in vitro DMS-treated piperidine fragmented genomic DNA (experimental control). Lanes 2 and 3 show the in vivo DMS-LMPCR generated products from unstressed and stressed (200 mM NaCl, 24 h) shoot samples, respectively. The distance of the respective bases from the +1 TSS is shown on the left side of the autoradiogram. The arrow heads on the right side of the autoradiogram show the sites of differential protein binding. An uncalibrated graph (drawn using ImageJ software) showing densitometric quantification of bands in each lanes is presented besides the autoradiogram. DNA sequence of the top strand corresponding to different regions of the autoradiogram has also been depicted. The underlined ‘G’ residues are the sites showing differences in the footprinting pattern and correspond to the arrow heads. (B) In vivo footprinting of the bottom strand. All representations are same as mentioned for (A)., ‘C’ residue in the top strand are marked in place of ‘G’ residues as the footprinting was done for the bottom strand. (C) Schematic representation of the cis-elements showing differential binding of transcription factors under control and stressed conditions (indicated by underlined ‘G’ and ‘C’ residues). The possible cis-elements have been represented by shaded boxes with their names. The location of TATA-Box, TSS and the third primer (R3 and F3) used in LMPCR are also shown. The number at the start of each sequence line is the base pair distance from the first TSS (+1 site).

Figure 8. EMSA with nuclear extracts from untreated and salt stressed plants.

(A) EMSA with a DNA oligo corresponding to a portion of DF4 fragment containing a SORLIP1 site, a SITE II like element and a telo-box. (B) EMSA with a DNA oligo containing AGCCCA element. (C) EMSA with a DNA oligo containing GGCCCA element. Lane 1 of (A), (B) and (C) represents the experimental control lane containing no protein. Lanes 2 and 3 include nuclear extracts from untreated and stressed (200 mM NaCl, 24 h) plants, respectively. The sequence of the double stranded oligos along with their relative position in the promoter with respect to the +1 TSS is mentioned beside each autoradiogram and the cis-elements are marked. The arrows indicate the gel shift bands and the star indicates the free probes.